Abstract: Large cerebral vessel occlusion is the most disabling and life-threatening form of ischemic stroke. Human stroke primarily occurs in late middle age and beyond. Approximately two thirds eligible patients treated with tPA experience incomplete reperfusion. Thrombectomy is now also a standard of care for treatment of acute stroke with large vessel occlusion. However, recanalization of the occluded large vessels by thrombectomy only leads to ~71% of patients achieving improved tissue reperfusion, often incomplete. In addition, due to unfavorably large ischemic cores, many patients with large artery occlusion are not eligible to receive tPA or thrombectomy. Patients with reperfusion of the ischemic tissue are closely associated with good clinical outcome. Thus, there is a compelling need to develop therapies in combination with tPA and thrombectomy to enhance cerebral perfusion and thereby augment the therapeutic efficacy of tPA and thrombectomy monotherapies. Also, therapies to block ischemic core expansion will increase numbers of patients who would be eligible to receive tPA and thrombectomy. Using rat models of embolic middle cerebral artery occlusion (eMCAO) and transient MCAO (tMCAO, ischemia/reperfusion), we found that exosomes derived from cerebral endothelial cells (CEC- exos) in combination with tPA after eMCAO or CEC-exos given upon reperfusion after tMCAO substantially increased recanalization and downstream cerebral blood flow (CBF), and reduced blood brain barrier (BBB) leakage and infarction compared to tPA or tMCAO alone. Exosomes are nano-vesicles that contain lipids, proteins, and RNAs including microRNAs (miRs). Our preliminary data suggest that exosomal cargo miRs likely contribute to the therapeutic effect of CEC-exos in combination with tPA on acute stroke by acting on cerebral endothelial cells to suppress proteins that promote thrombosis and BBB disruption. We thus propose to develop CEC-exo therapy as an adjunctive treatment to enhance tPA and thrombectomy treatments of acute ischemic stroke. Aim 1 is to investigate whether the CEC-exo therapy as an adjunctive treatment enhances tPA and thrombectomy treatments in aged rats after large artery occlusion. Aim 2 is to investigate whether CEC exosomal cargo miRs contribute to CEC-exos-amplified thrombolysis leading to reduction of neurovascular damage. Aim 3 investigates whether a special set of CEC-exo cargo miRs contribute to the therapeutic effect CEC-exos on stroke- induced neurovascular damage by suppressing a network of pro-BBB leakage and thrombotic genes. Accomplishing these aims will potentially lead to development of a mechanistically based exosome therapy as an adjunctive treatment to enhance tPA and thrombectomy treatments of acute ischemic stroke, leading to improvement in the neurological outcome.